Meda Surdokaité

“Nile Red” is a fluorescent, fat-soluble organic dye used in fluorescence microscopy for the selective identification of microplastics and in biological research for the localization and quantification of lipids in cells. It is therefore used a lot for methods of dyeing. The commercial price of microscopy grade “Nile Red” is approximately 150 EUR per 100 mg. The classical synthesis method for “Nile Red” is based on the use of N,N-dimethylformamide (DMF). As restrictions on the use of DMF solvent were approved on 19 November 2021 and are to be applied EU-wide from 2024, it is therefore vital to optimize a method based on another solvent that replace DMF in the synthesis of “Nile Red”, while maintaining an optimal cost for the compound.

Carried out nitrosation reaction of 3-diethylaminophenol to give 5-(diethylamino)-2-nitrosophenol, and intermediate product of reaction. This then can be dissolved with 1-naphthol in organic solvent. For the optimization of the “Nile Red” synthesis, different classes of organic solvents were selected. After that, the most promising organic solvent class was optimized. “Nile Red” purification and confirmation on chemical structure was performed.

Research provides 42% higher HPLC yield in multigram “Nile Red” synthesis by changing reaction environment from N,N-dimethylformamide to 2-propanol. “Nile Red” purity of 94%, suitable for the application in fluorescence microscopy, was achieved. It was then applied by the use for staining of cytoplasmic fat droplets, accumulated in cells.